Vero cells (green African monkey kidney epithelial cells, ATCC) were cultured in DMEM containing 4.5 g/L glucose (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S) at 37°C in 5% CO2. Expi293F cells were cultured in serum-free Expi293 Expression Medium at 37°C in 8% CO2. The infectious clone-derived DENV2 WT (GenBank accession: EU081177) and NS1:T164S mutant viruses used in this study were described previously (Chan et al., 2019 (link)). The anti-NS1 56.2 IgG antibody (Ab56.2) was obtained from the hybridoma cell culture as described previously (Rozen-Gagnon et al., 2012 (link)). Briefly, the Ab56.2 hybridoma cells were grown in PFHM II medium (Gibco) at 37°C in 5% CO2. Culture supernatants were collected every 4 d, clarified, and filtered through 0.2 μm filter membrane. Ab56.2 was purified from the supernatant through a Protein G HiTrap column (GE Healthcare) using the AKTA purification system (Cytiva). The bound Ab was eluted from the Protein G column using 0.1 M glycine (pH 2.7), neutralized with 1 M Tris-HCl (pH 9.0), and dialyzed with PBS for storage at –30°C until use. Cell lines were tested negative for mycoplasma contamination.
Akta purification system
Manufactured by Cytiva
Sourced in United Kingdom
The AKTA purification system is a versatile liquid chromatography platform designed for the purification and analysis of biomolecules. It provides precise control and monitoring of key parameters such as flow rate, pressure, and conductivity during the purification process. The AKTA system is a modular platform that can be configured with a range of columns, detectors, and other accessories to meet the specific requirements of various research and production applications.
Automatically generated - may contain errors
Lab products found in correlation
Benzonase, by Merck Group (3 mentions)
Thermomixer, by Eppendorf (3 mentions)
Benchtop cooling centrifuge, by Eppendorf (2 mentions)
Kanamycin, by Merck Group (2 mentions)
Superdex 75 column, by Cytiva (2 mentions)
Chicken egg lysozyme, by Thermo Fisher Scientific (2 mentions)
Hitrap protein g column, by GE Healthcare (1 mentions)
Pfhm 2 medium, by Thermo Fisher Scientific (1 mentions)
Dmem containing 4.5 g l glucose, by Thermo Fisher Scientific (1 mentions)
Immobilized papain resin, by Thermo Fisher Scientific (1 mentions)
Sephadex g 25 pd 10 column, by GE Healthcare (1 mentions)
Protein a hi trap column, by GE Healthcare (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Innova 44r shaking incubator, by Eppendorf (1 mentions)
Cary eclipse spectrofluorometer, by Agilent Technologies (1 mentions)
Chirascan plus spectropolarimeter, by Applied Photophysics (1 mentions)
Cary 60 spectrometer, by Agilent Technologies (1 mentions)
Electroporator, by Eppendorf (1 mentions)
Gel scanner, by Epson (1 mentions)
Low molecular weight markers, by Cytiva (1 mentions)
9 protocols using akta purification system
1
DENV2 WT and NS1:T164S Mutant Virus Production
2
Papain Digestion and Fab Purification
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1.8 mg of the purified Ab56.2 was subjected to papain digestion using the immobilized papain resin (Thermo Fisher) at an enzyme:substrate ratio of 1:160 according to the manufacturer’s instructions. The IgG-papain mixture was incubated at 37°C for 3.5 hr followed by removal of the immobilized papain resin to stop the digestion. The papain IgG digest was buffer-exchanged to PBS using PD10 Sephadex G25 column (GE Healthcare). The resulting Fab (Fab56.2) was then purified from the crude digest by Protein A HiTrap column (GE Healthcare) using the AKTA purification system (Cytiva), concentrated using 3 kDa MWCO Amicon concentrator (Millipore, Merck) and stored at –30°C until use.
3
Isolating Dengue NS1 Protein Complexes
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10 µg of isNS1wt or isNS1ts was complexed with papain-digested Fab56.2 or Ab56.2 at a protein:Fab or protein:Ab molar ratio of 1:5 by incubation for at least 2 hr on ice. 10 µg of isNS1wt, isNS1ts, or papain-digested Fab56.2 as well as the isNS1wt: or isNS1ts:Fab56.2 complex was then independently injected onto a Superdex 200 increase 3.2/300 GL column (GE Healthcare) connected to the AKTA purification system (Cytiva) in PBS (pH 7.4) at a constant flow rate of 0.075 mL/min. Chromatograms were analyzed on Unicorn 7 and replotted on OriginPro, version 2021b.
4
Protein A Purification of NHP IgG
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Pooled sera from NHP M16912 (2 mL total) was added to ∼30 mL of Protein A binding buffer and purified on a Protein A column on the AKTA purification system (Cytiva). Fractions 1A5-1A11 were pooled after Protein A purification. The second smaller peak was also pooled for analysis (1B1-1B9) (peak 2). Peak 1 Total IgG yield = 24.914 mg in 700ul at 35.592 mg/ml, Peak 2 total IgG yield = 1.726 mg in 190 ul at 9.084 mg/ml.
5
Affinity Chromatography Purification of ZIKV-EDIII
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The protein purification was performed by affinity chromatography using a 5 mL HisTrap® Fast Flow Crude prepacked column (Cytiva, Marlborough, MA, USA) coupled to the AKTA purification system (Cytiva, Marlborough, MA, USA). The column was equilibrated with a binding buffer (20 mM sodium phosphate, 500 Mm NaCl, 20 mM imidazole, pH 7.4), and the same binding buffer was mixed with an equal volume of the supernatant collected from the culture, injected into the equipment, and analyzed at a flow rate of 5 mL/min. The column-bound protein fraction was recovered after applying an elution buffer (20 mM sodium phosphate, 500 mM NaCl, 300 mM imidazole, pH 7.4). The purified protein was concentrated by ultrafiltration using an Amicon® Ultra Centrifugal Filter (Merck Millipore, Darmstadt, HE, Germany) and quantified by densitometry using ImageJ® software (https://imagej.nih.gov/ij/ accessed on 6 June 2021). For this, a standard curve of BSA was performed in the following concentrations: 250 µg/mL, 125 µg/mL, 62.5 µg/mL, 31.25 µg/mL, and 15.625 µg/mL. The densitometry values of each protein were used to construct an analytical curve, obtaining its equation, which was used to estimate the concentration of purified ZIKV-EDIII.
6
Purification and Characterization of MERS-CoV PL-pro
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The ORF of MERS-CoV PLpro (1484–1800 polyprotein residues, GenBank accession number NC_019843.2) was cloned into pET28a plasmid under T7 promoter as published before (Lin et al. 2014 (link)). The codon was optimized (GenScript, USA) and cloned between NcoI and XhoI sites which was in frame of C-terminal His tag present on the vector. E. coli BL21 (DE3) pLysS was used for the expression of recombinant protein. Low-molecular weight protein markers, prepacked Ni-NTA, and Superdex 75 columns were from Amersham Biosciences (United Kingdom). Chicken egg lysozyme was from USB Corporation, USA. Benzonase, ANS, and kanamycin from Sigma. IPTG was purchased from Bio Basic, Canada. All other chemicals used in this study were of reagent grade. Cary 60 spectrometer and Cary Eclipse spectrofluorometer were from Agilent technologies, USA. AKTA purification system was from Amersham Biosciences (United Kingdom) and SDS-PAGE assembly from Bio-Rad (USA). Thermomixer and benchtop cooling centrifuge were from Eppendorf, Germany. Innova 44R Shaking incubator was from New Brunswick, Germany. Chirascan-Plus spectropolarimeter was from Applied photophysics, United Kingdom.
7
Expression and Purification of cHSPA6
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The ORF of cHSPA6 cloned on pET15 vector and expression host E. coli BL21 (DE3) pLysS were kindly provided by Elrobh et al. (2011) (link). IPTG and ampicillin were obtained from Biobasic. Benzonase was purchased from Sigma, Chicken egg lysozyme from USB Corporation. Superdex 75, Ni–NTA resin, low molecular weight markers and prepacked columns were from Amersham Biosciences. All other chemicals used in this study were of reagent grade. Ultrospec 2100 pro Spectrophotometer, AKTA purification system, SDS–PAGE assembly were from Amersham Biosciences. Thermomixer, electroporator and benchtop cooling centrifuge were from Eppendorf. Lamp sterilizer from Cole-Parmer, shaking incubator from Jeio Tech, South Korea, gel scanner from Epson and pH meter was from Sentron.
8
Purification of Ageritin and Quinoin
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Ageritin and quinoin were purified according to previously reported procedures [1 (link),9 (link)]. Briefly, raw extracts from C. aegerita fruiting bodies or C. quinoa seeds were acidified with acetic acid and subjected to two consecutive chromatographic steps: Streamline SP (Cytiva, Bucinasco (MI), Italy) step-wise; gel-filtration by Sephadex G-75 Hi-load 26/60 column (Cytiva) on an Akta purification system (Amersham Pharmacia; Milan, Italy). Finally, the last step for ageritin purification is a low-pressure cation exchange chromatography step on a SP-Sepharose column (Cytiva) eluted with a NaCl gradient, while the last step for quinoin purification is a low-pressure cation exchange chromatography step on a CM-Sepharose column (Cytiva) eluted with a NaCl gradient.
Fractions corresponding to main peaks of ageritin or quinoin with inhibitory activity on cell-free protein synthesis were checked by SDS-PAGE analysis, pooled, dialyzed against water, freeze-dried, and stored at −20 °C until use.
Fractions corresponding to main peaks of ageritin or quinoin with inhibitory activity on cell-free protein synthesis were checked by SDS-PAGE analysis, pooled, dialyzed against water, freeze-dried, and stored at −20 °C until use.
9
MERS-CoV PLpro Protein Expression
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Open-reading frame 1 of the MERS-CoV PLpro, which encoded on the polyprotein1a, was cloned into a pET28a plasmid. The nucleotide sequence is optimized to enhance transcriptional and translational levels under the control of a strong T7 promoter (Lin et al. 2014 (link)). The codon was cloned between NcoI and XhoI in the C-terminal His tag of the vector using Genescript. Isopropyl β-D-1-thiogalactopyranoside (IPTG) was obtained from GenScript, and E. coli DE3-pLysS was purchased from EMD4 Biosciences (San Diego, CA, USA). Glycerol and NaCl were purchased from Scharlau Chemie, S.A. (Sentmenat, Spain); chicken egg lysozyme was obtained from Honeywell Research Chemicals (Morris Plains, NJ, USA); ANS, kanamycin, and benzonase was obtained from Sigma Aldrich (St. Louis, MO, UNA). The AKTA purification system and Superdex 75 columns were obtained from Amersham Biosciences (St. Giles, UK). PAGE 4–12 % bis-tris-gel was obtained from Life Technologies (Pleasanton, CA, USA); prepacked Ni-NTA columns were purchased from GE Healthcare. Agilent Technologies (USA) supplied the Cary 60 UV–vis spectrometer. The New Brunswick Innova 44R shaking incubator and Thermomixer were obtained from Eppendorf (Hamburg, Germany).
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